Single-cell suspensions from rhesus macaque tissues were used as input into the LEGENDScreen assay (Biolegend). Cell suspensions were incubated with Fc block (Human TruStain FcX, Biolegend) prior to labeling with fluorescently conjugated antibodies. To enable assessment of assay markers on cell derived from individual tissues, cells from bone marrow, lymph node, and PBMC were each labeled with anti-NHP CD45 (clone D058-1283, BD Biosciences) conjugated to a unique fluorescent tag to enable sample multiplexing and unmixing during analysis. Following labeling with CD45, samples were washed 2× with FACS buffer before being stained with antibodies targeting CD3 (SP34-2, BD Biosciences), CD20 (2H7, Biolegend), and CD138 (DL101, Biolegend). 1 × 106 cells were used as input into each well of the LEGENDScreen assay. Samples were acquired on a Symphony A5 (BD Biosciences) using FACSDiva (BD Biosciences).
Legendscreen assay
Manufactured by BioLegend
LEGENDScreen assay is a multiplex cell-based screening platform developed by BioLegend. It allows for the simultaneous detection and quantification of multiple protein targets in a single sample.
Automatically generated - may contain errors
3 protocols using legendscreen assay
1
Multiplexed Rhesus Macaque Cell Profiling
2
Comprehensive Multiparameter Cell Profiling
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A panel of 124 oligo-conjugated antibodies containing 115 antibodies targeting surface-expressed proteins of interest and 9 isotype controls was used in these studies. All antibodies, except 1, were in the TotalSeqA format and available commercially (Biolegend). CD38-bio (clone OKT10, Caprico Biotechnologies) and oligo-conjugated streptavidin-PE (SA-PE) (Biolegend) were used to detect CD38 in this panel. Details on antibodies used and clone information are contained in Supplementary Data 2 . All antibodies used were determined to be cross-reactive between human and rhesus using the LEGENDScreen assay (Biolegend).
3
Multiparameter Flow Cytometry Immunophenotyping
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Cell suspensions were stained with the antibodies indicated (Table S3) in Brilliant stain buffer (BD Biosciences) containing 4% normal mouse serum according to standard techniques. The cells were stained with 7-AAD and analyzed on an LSR Fortessa 2 (BD Biosciences) using Flowjo software (Treestar).
For the Legendscreen assay (Biolegend), each antibody was first resuspended in 35 µl of FACS buffer (PBS with 5% FCS and 0.05% sodium azide). Up to 300x10 6 cells were stained with the antibody backbone panel (Table S4), washed and resuspended in PBS with 5% FCS. 40 µl of cell suspension was aliquoted into each well of V-bottom 96-well plates containing 10 ul of the PEconjugated antibody per well. The cells were analysed using an LSR Fortessa 2 with High Throughput Sampler (BD).
For the Legendscreen assay (Biolegend), each antibody was first resuspended in 35 µl of FACS buffer (PBS with 5% FCS and 0.05% sodium azide). Up to 300x10 6 cells were stained with the antibody backbone panel (Table S4), washed and resuspended in PBS with 5% FCS. 40 µl of cell suspension was aliquoted into each well of V-bottom 96-well plates containing 10 ul of the PEconjugated antibody per well. The cells were analysed using an LSR Fortessa 2 with High Throughput Sampler (BD).
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